Sleep-promoting peptides and their uses

Abstract

A sleep-promoting peptide and a composition comprising the sleep-promoting peptide are provided. The peptide and the composition have a use for promoting sleep in a subject. Specific peptides are described in sequence identification numbers 1 to 139.

Description

Technology Field

[0001] The present invention relates to a peptide and a composition comprising said peptide. The peptide of the present invention has sleep-promoting activity. The present invention also relates to the use of the peptide and the composition of the present invention. Background Technology

[0002] Sleep is essential for human health and well-being. The importance of sleep as a risk factor for poor health is often overlooked. Sleep deprivation can have detrimental effects on cognition, anxiety, mental health, energy levels, and cardiovascular and metabolic health. Beyond individual health, the quantity and quality of sleep can affect work performance, relationships, and accidents on the road, at work, or at home, and therefore represent a risk to public health and safety (Ramar, Malhotra, Carden et al., (2021) J Clin Sleep Med, 17(10), pp. 2115-2119).

[0003] Sleep quality can be affected by countless factors ranging from untreated sleep disorders to lifestyle habits and certain medications. However, one of the most common factors affecting sleep is stress.

[0004] Stress has become a serious social issue and can have a dual impact on sleep. Lifestyle stressors, such as financial, work pressure, or family problems, often disrupt the process of falling asleep and can shorten and fragment periods of rest, which have cascading effects on brain health, memory, and cellular recovery. Furthermore, sleep loss can trigger the acute activation of the endogenous stress response system and stress hormones, such as cortisol, which not only impairs sleep quality but can also accelerate metabolic and cognitive outcomes over time (Leproult, Copinschi, Buxton et al., (1997) Sleep 20(10):865-70.). Cortisol is a steroid hormone produced by the adrenal glands located above the kidneys; it promotes arousal and rises during periods of stress.

[0005] The transition from wakefulness to sleep is based on several mechanisms, human circadian rhythms, and sleep-wake homeostasis. Human circadian rhythms operate on a 24-hour basis and stimulate many of the body's daily functions, including body temperature, metabolism, and hormone release. This enables the body to initiate sleep by evoking a sense of fatigue through the activation and / or inhibition of key hormones and neurotransmitters, including, for example, norepinephrine, serotonin, acetylcholine, histamine, dopamine, and orexin (Valentio & Volkow (2019) Nature Publishing Group, pp. 1-3). Sleep-wake homeostasis mechanisms recognize internal and external influences that can affect an individual's sleep, such as stress, nutrition, medical conditions, or exposure to light.

[0006] There are various sleep aids and sleep medications available on the market, either by prescription or over-the-counter. These range from small molecules to natural ingredients, and some of them target known sleep-promoting pathways, such as γ-aminobutyric acid (GABA) and melatonin receptors, histamine-1 receptors, and orexin receptors. Some of the small molecule ingredients fall into one of four categories: (1) non-benzodiazepines, e.g., eszopiclone and diphenhydramine or DHH, (2) benzodiazepines, e.g., flurazepam, (3) melatonin receptor agonists, e.g., rameltron, and (4) orexin receptor antagonists, e.g., suvorexant.

[0007] Importantly, the medications listed above should be taken only for short periods due to the potential for dependence, habituation, and tolerance. Additional undesirable side effects associated with prescription sleep aids include headache and drowsiness. One of the greatest limitations of prescription medications is that while they promote sedation and unconsciousness, they fail to induce rapid eye movement (REM) sleep, the most restorative stage of sleep (Newman & Alschuler (2022) Journal of Interprofessional Education & Practice 29:100569.).

[0008] Another limitation associated with these drugs is their potential to enhance the sedative effects of inhibitors and / or other drugs. Because clinical studies in this complex area are limited, further investigation is required into potential complications associated with the long-term use of prescribed sleep medications.

[0009] Orexin is a neurotransmitter that regulates various physiological functions, including the sleep-wake cycle, appetite, memory, and stress response (Wang et al., (2018) Frontiers in Molecular Neuroscience. Frontiers Media SA). It has been a successful target for sleep medications, such as the small molecule orexin receptor antagonist suvorexant, which have been suggested to suppress abnormal sleep behaviors. Unfortunately, small molecule therapies are notorious for being associated with off-target effects and unfavorable safety profiles. Furthermore, the high cost of suvorexant makes it unaffordable for many people suffering from sleep disturbances.

[0010] Prescription sleep aids are commonly prescribed; however, they can cause undesirable side effects, such as headache, drowsiness, tolerance, and dependence. One of the biggest limitations of these medications is that while they promote sedation and unconsciousness, they fail to induce rapid eye movement (REM) sleep, the most restorative stage of sleep (Newman & Alschuler (2022) Journal of Interprofessional Education & Practice 29:100569.).

[0011] Natural alternatives such as melatonin, ashwagandha, valerian, and magnesium exist, some of which are the most studied. However, there is a lack of high-quality research and scientific consensus regarding the efficacy and safety of many natural sleep aids (Guadagna, Barattini, Rosu et al., (2020) Evid Based Complement Alternat Med. 2020:3792390).

[0012] The lack of effective solutions makes the search for natural, safe, and proven nutritional interventions that promote relaxation, reduce stress, and foster healthy sleep an urgent unmet need.

[0013] The present invention aims to alleviate one or more problems of the prior art.

[0014] The present invention provides a plant-based solution that reduces stress, promotes tranquility outside the brain, and bypasses additional pathways. The present invention provides one or more bioactive peptides that act as antagonists of orexin receptors. Inhibition of these receptors reduces cortisol release and thereby induces a relaxed state that enables sleep.

[0015] The inventors have discovered a peptide that promotes sleep in subjects. The peptide reduces peripheral cortisol concentrations by targeting the orexin pathway and decreasing the concentration of peripheral cortisol released from the adrenal glands. By modulating the orexin signaling pathway and reducing circulating levels of cortisol, the present invention reduces stress, promotes relaxation, and enables the initiation of natural sleep cycles and cellular recovery processes in a safe and non-addictive manner.

[0016] When tested in adrenal cortical cells in an in vitro environment, the peptide of the present invention was shown to antagonize orexin signaling, which resulted in a significant absolute decrease of 17.4% in cortisol secretion and a 44.3% decrease in induced cortisol (Figs. 2 to 4).

[0017] The peptide of the present invention also has important auxiliary functions for sleep, such as anti-inflammatory and anti-anxiety effects. Therefore, the present invention has a multi-mechanism approach for reducing stress and having a positive effect on sleep.

[0018] An aspect of the present invention provides a peptide (hereinafter referred to as the “peptide of the present invention”) having a length of typically up to 50 amino acids, comprising (or consisting of) an amino acid sequence selected from sequence identification numbers: 1 to 5 and 97 to 100, or a variant of the amino acid sequence.

[0019] The peptide of the present invention is derived from brown rice (Oryza sativa).

[0020] The amino acid sequences of sequence identification numbers 1 to 6 and 97 to 100 are as follows:

[0021] Sequence Identification Number: 1: YYPGLSN

[0022] Sequence Identification Number: 2: VSEWDPSKDK

[0023] Sequence Identification Number: 3: QGLLVPRYSNTPGLVY

[0024] Sequence Identification Number: 4: GATDVGHPM

[0025] Sequence Identification Number: 5 FATPPVHGS

[0026] One example of a variant of sequence identification number: 4 is sequence identification number: 6.

[0027] Sequence identification number: 6 has the following amino acid sequence.

[0028] Sequence Identification Number: 6: ATDVGHPM

[0029] Sequence Identification Number: 97 QLLSQSTS

[0030] Sequence Identification Number: 98 QQQYYPGLS

[0031] Sequence Identification Number: 99: LADTYNPR

[0032] Sequence Identification Number: 100: IGGIGTVPVGR

[0033] The peptide of the present invention is a sleep-promoting peptide.

[0034] In an embodiment, the peptide of the present invention has anti-inflammatory activity.

[0035] In an embodiment, the peptide of the present invention has anti-anxiety activity.

[0036] In an embodiment, the peptide of the present invention is biologically active.

[0037] In an embodiment, the peptide of the present invention is a modified peptide. In an embodiment, the peptide is modified to increase its lipophilicity. In an embodiment, the peptide is modified to increase its half-life. In an embodiment, the N-terminal or C-terminal amino acid of the peptide is modified. In an embodiment, the peptide is modified to a protecting group. In one embodiment, the N-terminal or C-terminal amino acid of the peptide is modified to a protecting group.

[0038] An aspect of the present invention provides a conjugate (referred herein to be “conjugate of the present invention”) comprising one or more peptides of the present invention that are conjugated to, linked to, or fused to a binding partner. In an embodiment, the peptides of the present invention are modified with a reactive group or similar that is configured to allow conjugation to a binding partner.

[0039] An aspect of the present invention provides a composition comprising one or more peptides of the present invention (referred to herein as "composition of the present invention"). The composition of the present invention may comprise at least two peptides of the present invention, preferably three or more peptides of the present invention, preferably four or more peptides of the present invention, and preferably five or more peptides of the present invention.

[0040] The composition of the present invention may comprise a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 1, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 2, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 3, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 4, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 5, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 5, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 97, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 98, a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 99, and / or a peptide or variant thereof comprising (or consisting thereof) sequence identification number: 100.

[0041] In an embodiment of the present invention, the composition comprises one or more peptides selected from peptides comprising (or composed thereof) sequences selected from sequence identification numbers: 1 to 5.

[0042] In an embodiment of the present invention, the composition comprises one or more peptides selected from peptides consisting of sequences selected from sequence identification numbers: 1 to 5 and 97 to 100.

[0043] In an embodiment of the present invention, the composition comprises all peptides of the present invention.

[0044] In an embodiment, the composition of the present invention is a powder. The powder is a food-grade powder, that is, suitable for addition to food or feed products or beverage products.

[0045] In an embodiment, the composition of the present invention is formulated as a capsule. In an embodiment, the capsule comprises the powder of the present invention. The capsule is food grade, i.e., suitable for addition to food, beverage, or feed products.

[0046] In an embodiment, the powder comprises one or more peptides of the present invention (w / w) in an amount of about 0.0001 to about 1.0%, about 0.0001 to about 0.2%, about 0.001 to about 0.1%, or about 0.01 to about 0.1%.

[0047] In an embodiment, the composition is edible (edible). In one embodiment, the composition is a food or beverage. It is suitable for consumption by humans, animals (mammals), or birds, preferably for human consumption.

[0048] In an embodiment, the composition is a nutritional or dietary supplement.

[0049] In an embodiment, the composition is a pharmaceutical composition comprising optionally at least one pharmaceutically acceptable excipient.

[0050] Preferably, the composition is artificial.

[0051] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in promoting sleep in a subject. This may involve the induction of sleep in the subject.

[0052] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in improving sleep in a subject.

[0053] "Improving sleep" may include maintaining sleep cycles, such as maintaining appropriate durations for sleep cycles including rapid eye movement (REM) and deep sleep.

[0054] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in inducing relaxation in a subject. This may include the induction of equilibrium in a subject.

[0055] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in reducing or preventing stress in a subject.

[0056] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in reducing cortisol in a subject.

[0057] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in promoting recovery from fatigue in a subject. In an embodiment, recovery is after infection in the subject.

[0058] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in reducing or preventing anxiety in a subject.

[0059] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in the treatment or prevention of inflammation in a subject.

[0060] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in the treatment or prevention of inflammatory disorders in a subject.

[0061] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in increasing concentration in a target. "Concentration" may include an increase in the range of attention and / or productivity in the target.

[0062] An aspect of the present invention provides a peptide of the present invention or a composition of the present invention for use in improving cognitive function or ability in a subject.

[0063] An aspect of the present invention provides a non-therapeutic method for treating or preventing inflammation in a subject, comprising administering a peptide of the present invention or a composition of the present invention to a subject. Examples of non-therapeutic treatments for inflammation include uses for alleviating normal, non-pathological inflammation.

[0064] An aspect of the present invention provides a non-therapeutic method for promoting or improving sleep in a subject, comprising administering a peptide of the present invention or a composition of the present invention to a subject.

[0065] In one aspect, the present invention provides a nucleic acid encoding one or more peptides of the present invention.

[0066] In one aspect, the present invention provides an expression vector comprising DNA encoding one or more peptides of the present invention, wherein the vector is configured for heterogeneous expression of one or more peptides of the present invention in a host cell (hereinafter "expression vector of the present invention").

[0067] In an aspect, the present invention provides a host cell, particularly a bacterial or mammalian producer cell (hereinafter "transformed cell of the present invention"), engineered to heterogeneously express one or more peptides of the present invention. In one embodiment, the transformed host cell comprises an expression vector of the present invention.

[0068] The present invention also provides a method for producing one or more peptides of the present invention, comprising the steps of providing a transformed cell of the present invention, culturing the transformed host cell to induce heterogeneous expression of the recombinant peptide of the present invention by the host cell, and recovering the recombinant peptide of the present invention.

[0069] The present invention also provides a method for manipulating cells for heterogeneous expression of one or more peptides of the present invention, comprising the step of transforming cells with an expression vector of the present invention, wherein the cells transformed thereby are capable of heterogeneous expression of one or more peptides of the present invention.

[0070] definition

[0071] All published documents, patents, patent applications, and other references mentioned herein are incorporated herein by reference in their entirety for all purposes, as specifically and individually indicated, whereby each individual published document, patent, or patent application is incorporated by reference and its contents are cited in their entirety.

[0072] As used herein and unless specifically otherwise indicated, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings that the terms may enjoy in the relevant technical field:

[0073] Unless otherwise required by the context, the use of the singular form herein shall be interpreted to include the plural form and vice versa. Singular terms used in relation to entities shall be interpreted to refer to one or more such entities. Accordingly, singular terms, “one or more,” and “at least one” are used interchangeably herein.

[0074] The term “comprising,” or variations thereof such as “comprising” or “comprising,” as used herein, shall be interpreted as indicating that any mentioned completeness (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of completenesses (e.g., features, elements, characteristics, property, method / process steps, or limitations) is included, but does not exclude any other completeness or group of completenesses. Accordingly, the term “comprising,” as used herein, does not exclude any additional, unmentioned completenesses or method / process steps, whether comprehensive or open-ended.

[0075] As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to include any disorder, disease, abnormality, pathology, illness, condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology (or whether an etiological basis for the disease is actually established). This therefore includes conditions arising from infection, trauma, injury, surgery, radiation therapy, poisoning, or nutritional deficiency.

[0076] As used herein, the terms “treatment” or “treating” refer to an intervention (e.g., administration of an agent to a subject) that cures, improves, or alleviates the symptoms of a disease, or eliminates the cause(s) thereof (or reduces their effects). In this case, the term is used synonymously with the term “therapy.” This may be manifested by a permanent or temporary improvement in the subject’s condition. In this context, this includes limiting or reversing the progression of the disease.

[0077] The terms “prevention” or “preventing” as used herein refer to an intervention (e.g., administration of an agent to a subject) that prevents or delays the onset or progression of a disease or the severity of the disease in a subject, or reduces (or eradicates) its incidence within a treated population.

[0078] As used herein, or as applied to reference peptides or compositions, the term "therapeutic effective dose" or "effective dose" means an amount that can be administered to a subject without excessive toxicity, irritation, allergic reaction, or other problems or complications in accordance with a reasonable benefit-risk ratio, but is sufficient to provide the desired effect, such as treatment or prevention manifested by permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject depending on the individual's age and overall condition, the mode of administration, and other factors. Therefore, while it is not possible to specify an exact effective dose, a person skilled in the art will be able to determine an appropriate "effective" dose for any individual case using routine experimentation and background knowledge. In this context, treatment outcomes include the eradication or alleviation of symptoms, reduced pain or discomfort, long-term survival, improved mobility, and markers of other clinical improvements. Treatment outcomes do not need to be complete cures.

[0079] As used herein, the term “peptide” typically refers to a polymer composed of up to 50 amino acid monomers linked by peptide bonds. The peptide may be 3 to 50 amino acid lengths. The peptide may be 4 to 50 amino acid lengths. The peptide may be 5 to 50 amino acid lengths. The peptide may be 6 to 50 amino acid lengths. The peptide may be 7 to 50 amino acid lengths. The peptide may be 8, 9, or 10 to 50 amino acid lengths. The peptide may be up to 20 or 30 amino acid lengths. The peptide of the present invention and for use therein (including fragments and variants thereof) may be produced wholly or partially by chemical synthesis or by expression from nucleic acids. For example, the peptides of the present invention and for use therein can be easily prepared according to widely established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the relevant art (e.g., the literature [JM Stewart and JD Young, Solid Phase Peptide Synthesis, 2 nd [See M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984)]). If necessary, any of the peptides used in the present invention may be chemically modified to increase their stability. Typically, the peptides are linear peptides.

[0080] “Chemically modified peptide” or “peptide analog” comprises any functional or chemical equivalent of a peptide characterized by its increased stability and / or efficacy in vivo or in vitro in connection with the practice of the present invention. The term peptide analog also refers to any amino acid derivative of a peptide as described herein. Peptide analogs may be produced by procedures, including but not limited to modifications to side chains, the incorporation of non-natural amino acids and / or derivatives thereof during peptide synthesis, and the use of crosslinking agents, and other methods that impose stereotype constraints on the peptide or its analogs. Examples of side chain modifications include, for instance, reductive alkylation by reduction with NaBH4 following a reaction with an aldehyde; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of the amino group with a cyanate; and trinitrobenzylation of the amino group with 2,4,6,trinitrobenzenesulfonic acid (TNBS). It includes alkylation of the amino group using succinic anhydride and tetrahydrophthalic anhydride; and modification of the amino group by reduction using NABH4 following pyridoxylation of lysine using pyridoxa-5'-phosphate. The guanidino group of the arginine residue may be modified by the formation of a heterocyclic condensation product using reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal. The carboxyl group may be modified by carbodiimide activation through o-acylisourea formation, followed by, for example, subsequent derivatization to the corresponding amide.The sulfhydryl group may be modified by methods such as carboxymethylation using iodoacetic acid or iodoacetamide; oxidation of performic acid to cysteic acid; formation of disulfide mixed with other thiol compounds; maleimide; reaction with maleic anhydride or other substituted maleimide; formation of mercury derivatives using 4-chloromercurybenzoate, 4-chloromercuryphenylsulfonic acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercury; and carbamylation using cyanates at an alkaline pH. The tryptophan residue may be modified by, for example, oxidation using N-bromosuccinimide or alkylation of the indole ring using 2-hydroxy-5-nitrobenzyl bromide or sulfonyl halide. Tyrosine residues can be modified by nitration using tetranitromethane to form 3-nitrotyrosine derivatives. Modification of the imidazole ring of histidine residues can be achieved by alkylation using iodoacetic acid derivatives or N-carbethoxylation using diethylpyrocarbonate. Examples of incorporating non-natural amino acids and derivatives during peptide synthesis include, but are not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and / or D-isomers of amino acids. Peptide structural modification involves the generation of retro-inverso peptides containing inverted sequences encoded by D-amino acids.

[0081] In this specification, the term “sequence identity” should be understood to mean the amount of matching nucleotides between different sequences. For example, a 16S rRNA gene sequence that shares at least 98% sequence identity with a reference sequence is one in which any 98% of the aligned nucleotides of the variant are identical to the corresponding nucleotides in the reference sequence over the entire length of the sequence. Sequence identity is the amount of exactly matching characters between two different sequences. Thus, gaps are not counted, and the measurement relates to the shorter of the two sequences.

[0082] The term “variant” refers to a peptide that is substantially identical to the peptide of the present invention as defined herein, but is modified in relation to one or more amino acid residues. Preferably, such modification involves the insertion, addition, deletion, and / or substitution of six or fewer amino acids, preferably five or fewer, four or fewer, more preferably three or fewer, and most preferably only one or two amino acids. Insertions, additions, and substitutions using natural and modified amino acids are envisioned. The variant may have conservative amino acid changes that are structurally, chemically, or functionally similar to the amino acid being substituted. The term “variant” is also intended to include chemical derivatives of the protein, i.e., wherein one or more residues are chemically derivatized by the reaction of a functional sider. Additionally, variants are included in which naturally occurring amino acid residues are replaced by amino acid analogs. Details of amino acid analogs are widely known to those skilled in the art. The variant retains the activity of the peptide, such as sleep-promoting activity.

[0083] In the embodiments, the variant is a “therapeutic variant.” The term “therapeutic variant,” as applied to the reference peptide, means a peptide having substantially the same amino acid sequence as the reference peptide, which is therapeutically effective as defined herein, e.g., sleep-promoting activity, i.e., is a sleep-promoting variant. Accordingly, for example, the term should be understood to include a variant that is modified with respect to one or more amino acid residues as disclosed above. Generally, the variant will have at least 50%, 60%, or 70% amino acid sequence identity with the parent sequence, preferably at least 80% sequence identity, more preferably at least 90% sequence identity, and ideally at least 95%, 96%, 97%, 98%, or 99% sequence identity. It should be noted that any variant will have primarily the same therapeutic effect or an enhanced effect when tested in vitro or in vivo models of the disease.

[0084] The term “variant” is also understood to include the term “fragment” and accordingly refers to a segment of the peptide of the present invention. Typically, the fragment has a length of 3 to 15 adjacent amino acids. Typically, the fragment has a length of 4 to 12, or 5 to 10 adjacent amino acids. Generally, the fragment has a charge of about -1 to +1 at pH 7. The charge of the peptide, fragment, or region is determined using the method of the literature [Cameselle, JC, Ribeiro, JM, and Sillero, A. (1986). Derivation and use of a formula to calculate the net charge of acid-base compounds. Its application to amino acids, proteins and nucleotides. Biochem. Educ. 14, 131-136].

[0085] As used herein, the term “subject” is any subject requiring treatment or prevention. This may be a mammal, e.g., a human, or an animal. In an embodiment, this is a human subject.

[0086] "Mammal" means human or animal.

[0087] As used herein, the term “conjugate” refers to an embodiment of the present invention in which a peptide is conjugated, linked, or fused to a binding partner, e.g., one or more polyethylene glycol polymers or other compounds, e.g., a molecular weight-increasing compound or a lipophilic group. The molecular weight-increasing compound is any compound that will typically increase the molecular weight of the resulting conjugate by 10% to 90%, or 20% to 50%, and may have a molecular weight of 200 to 20,000, preferably 500 to 10,000. The compound that increases molecular weight may be PEG, any water-soluble (bipolar or hydrophilic) polymer moiety, homopolymers or copolymers of PEG, monomethyl-substituted polymers of PEG (mPEG) and polyoxyethylene glycerol (POG), polyamino acids such as poly-lysine, poly-glutamic acid, poly-aspartic acid, particularly those of the L stereotype, and pharmacologically inactive proteins such as albumin, gelatin, fatty acids, polysaccharides, lipid amino acids, and dextran. The polymer moiety may be straight-chain or branched and may have a molecular weight of 500 to 40000 Da, 5000 to 10000 Da, or 10000 to 5000 Da. The compound (binding partner) may be any suitable cell-penetrating compound, such as tat peptide, penetratin, or pep-1. The compound (binding partner) may be an antibody molecule. The compound (bonding partner) may be a lipophilic moiety or a polymeric moiety. Lipophilic substituents and polymeric substituents are known in the relevant art. Lipophilic substituents include acyl groups, sulfonyl groups, N atoms, O atoms, or S atoms, which form part of an ester, sulfonyl ester, thioester, amide, or sulfonamide. The lipophilic moiety may include a hydrocarbon chain having 4 to 30 C atoms, preferably 8 to 12 C atoms. It may be linear or branched, saturated or unsaturated. The hydrocarbon chain may be further substituted.This may be a cycloalkane or a heterocycloalkane. The peptide may be modified at the N-terminus, C-terminus, or both. The polymer or compound (bonding partner) is preferably connected to an amino, carboxyl, or thio group and may be connected by the N-terminus or C-terminus of the side chain of any amino acid residue. The polymer or compound (bonding partner) may be conjugated to the side chain of any suitable residue. The polymer or compound (bonding partner) may be conjugated via a spacer. The spacer may be a natural or non-natural amino acid, succinic acid, lysyl, glutamyl, asparagyl, glycyl, beta-alanyl, or gamma-aminobutanoyl. The polymer or compound (bonding partner) may be conjugated via an ester, sulfonyl ester, thioester, amide, carbamate, urea, or sulfonamide. A person skilled in the art knows suitable means for preparing the described conjugate.

[0088] In terms of "sequence homology," the term should be understood to mean a variant (or homolog) that shares a defined percentage similarity or identity with the reference sequence when the percentage of aligned residues of the variant (or homolog) is the same as the corresponding residues in the reference sequence, or is a conservative substitution thereof, and when the variant (or homolog) shares the same function as the reference sequence.

[0089] This alignment and percentage homology or sequence identity may be determined using software programs known in the relevant art; for example, one alignment program is BLAST, which uses default parameters. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi.

[0090] A variant (or homolog) that shares 70% sequence identity with a reference sequence is one in which any 70% of the aligned residues of the variant (or homolog) are identical to the corresponding residues in the reference sequence or are conservative substitutions thereof over the entire length of the sequence. Sequence identity is the amount of exactly matching characters between two different sequences. Thus, gaps are not counted, and the measurement is related to the shorter of the two sequences.

[0091] The term “natural” as applied to peptides means (a) a peptide comprising a fragment of a plant protein, typically a flavavin protein, or a variant of a flavavin protein. The peptide or fragment of the present invention may be isolated from a plant protein or synthesized.

[0092] The "C-terminal domain" as applied to the fragment refers to the first three amino acids at the c-terminus of the fragment.

[0093] The "N-terminal domain" as applied to the fragment refers to the last three amino acids at the n-terminus of the fragment.

[0094] The term "biological activity," as applied to peptides or fragments, means having a health-promoting effect when administered to mammals for sleep-promoting activity.

[0095] "Modified Peptide": In embodiments of the present invention, the peptide is a modified peptide. The term modified peptide is used interchangeably with derivatives of the term peptide. A modified peptide comprises a peptide substituted with one or more groups as defined herein. The modification may be any modification that provides the peptide and / or composition of the present invention with an increased ability to penetrate cells. The modification may be any modification that increases the half-life of the composition or peptide of the present invention. In one embodiment, the group is a protecting group. The protecting group may be an N-terminal protecting group, a C-terminal protecting group, or a side chain protecting group. The peptide may have one or more of these protecting groups. A person skilled in the art knows suitable techniques for reacting amino acids with these protecting groups. These groups may be added by manufacturing methods known in the art, for example, as outlined in paragraphs

[0104] through

[0107] of US2014120141. The group may remain on the peptide or may be removed. The protecting group may be added during synthesis. In embodiments of the present invention, the peptide may be substituted with one or more saturated or unsaturated groups selected from straight or branched chains, long chains or short chains, having 1 to 29 carbon atoms, which are substituted or unsubstituted with hydroxyl, amino, aminoacyl, sulfate, or sulfide groups. N-acyl derivatives comprise acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, octanoic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isosteric acid, elidoic acid, 2-ethylhexanoic acid, coconut oil fatty acids, tallow fatty acids, hydrogenated tallow fatty acids, palm kernel fatty acids, lanolin fatty acids, or similar acids. These may be substituted or unsubstituted. When substituted, they are preferably substituted with hydroxyl, or sulfur-containing groups such as (but not limited thereto) SO3H, SH, or SS. In an embodiment of the present invention, the peptide is R1-X-R2.The R1 and / or R2 groups are each attached to the amino-terminal (N-terminus) and carboxyl-terminus (C-terminus) of the peptide sequence. In one embodiment, the peptide is R1-X. Alternatively, the peptide is X-R2. Preferably, R1 is H, C. 1-4 It is alkyl, acetyl, benzoyl, or trifluoroacetyl. X is a peptide of the present invention; and R2 is OH or NH2. In an embodiment, R1 is selected from the group formed by H, a non-cyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, Tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), and R5-CO-, wherein R5 is selected from the group formed by H, a non-cyclic substituted or unsubstituted aliphatic group, a substituted or unsubstituted alicyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocyclyl, and a substituted or unsubstituted heteroarylalkyl;

[0096] R2 is selected from the group formed by -NR3R4, -OR3, and -SR3, wherein R3 and R4 are independently selected from the group formed by H, non-cyclic substituted or unsubstituted aliphatic groups, substituted or unsubstituted alicyls, substituted or unsubstituted heterocyclyls, substituted or unsubstituted heteroarylalkyls, substituted or unsubstituted aryls, and substituted or unsubstituted aralkyls; provided that R1 and R2 are not α-amino acids. According to another preferred embodiment, R2 is -NR3R4, -OR3, or -SR3, wherein R3 and R4 are H, substituted or unsubstituted C1-C 24 Alkyl, substituted, or unsubstituted C2-C 24 Alkenyl, Tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), substituted or unsubstituted C2-C 24alkynyl, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloalkinyl, substituted or unsubstituted C 6- C 30 Aryl, substituted or unsubstituted C7-C 24 Independently selected from the group formed by aralkyls, substituted or unsubstituted heterocyclyl rings of 3 to 10 members, and substituted or unsubstituted heteroarylkyls of 2 to 24 carbon atoms and 1 to 3 non-carbon atoms, wherein the alkyl chain is of 1 to 6 carbon atoms. Optionally, R3 and R4 may be joined by saturated or unsaturated carbon-carbon bonds to form a ring having a nitrogen atom. More preferably, R2 is -NR3R4 or -OR3, where R3 and R4 are H, substituted or unsubstituted C1-C 24 Alkyl, substituted, or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 alkynyl, substituted or unsubstituted C3-C 10 Cycloalkyl, substituted or unsubstituted C6-C 15It is independently selected from the group formed by aryl and 3-10 member substituted or unsubstituted heterocyclyls, and substituted or unsubstituted heteroarylkyls having a 3-10 member ring and an alkyl chain of 1-6 carbon atoms. More preferably, R3 and R4 are selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. Even more preferably, R3 is H and R4 is selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. According to a more preferred embodiment, R2 is selected from -OH and -NH2. According to another embodiment of the present invention, R1 is selected from the group formed by H, acetyl, lauroyl, myristoyl, or palmitoyl, and R2 is -NR3R4 or -OR3, wherein R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl, and hexadecyl, preferably R2 is -OH or -NH2. More preferably, R1 is acetyl or palmitoyl and R2 is -NH2. In a preferred embodiment, an acyl group is attached to the N-terminal end of at least one amino acid of the peptide. In an embodiment of the present invention, the peptide is modified to include a side chain protector. The side chain protector may be one or more of the group comprising benzyl or benzyl-based groups, t-butyl-based groups, benzyloxy-carbonyl (Z) groups, and allyloxycarbonyl (alloc) protectors. The side chain protector may be derived from a non-chiral amino acid, such as non-chiral glycine. The use of achiral amino acids helps stabilize the resulting peptide and also facilitates the easy synthetic pathway of the present invention. Preferably, the peptide further comprises a modified C-terminus, preferably an amidated C-terminus. The achiral residue may be alpha-aminoisobutyric acid (methylalanine). It will be recognized that the specific side chain protector used will depend on the sequence of the peptide and the type of N-terminal protector used.

[0097] The term "artificial," as applied to edible products, should be understood to mean something manufactured by humans and not found in nature.

[0098] "Dietary supplement" refers to a product suitable for mammalian consumption as a means of supplementing the main diet of mammals.

[0099] In this specification, the term "composition" should be understood to mean that which is produced by human effort and does not exclude naturally occurring compositions. Exemplary compositions include food, beverages, nutritional supplements, personal care compositions, and pharmaceutical compositions.

[0100] "Pharmaceutical composition": Further aspects of the present invention relate to a pharmaceutical composition comprising a peptide of the present invention or a composition of a peptide of the present invention mixed with one or more pharmaceutically acceptable excipients (including diluents and carriers). Although the peptide and composition of the present invention may be administered alone, they will generally be administered mixed with pharmaceutical excipients, particularly for human therapy. The pharmaceutical composition may be for human or animal use in human and veterinary medicine.

[0101] In embodiments of the present invention, excipients may be suitable diluents, carriers, binders, lubricants, suspending agents, coating agents, preservatives, stabilizers, dyes, vehicles, solubilizers, bases, softeners, emulsifiers, fragrances, wetting agents, and / or surfactants.

[0102] Examples of such suitable excipients for the various different forms of the pharmaceutical compositions described herein are found in the literature ["Handbook of Pharmaceutical Excipients, 2 ndIt can be found in [Edition, (1994), Edited by A Wade and PJ Weller]. In particular, formulations for topical delivery are described in the literature [Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis], the full contents of which are incorporated herein by reference.

[0103] Carriers or diluents accepted for therapeutic use are widely known in the pharmaceutical technology field and are described, for example, in the literature [Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985)].

[0104] Examples of suitable diluents include, but are not limited to, any diluent disclosed in US2014120131 or US2004132667. Examples include ethanol, glycerol, and water.

[0105] Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc., and any suitable carrier disclosed in US 2014120131 or US 2004132667. In some embodiments, the carrier may include, but is not limited to, a liquid, such as water, an oil, or a surfactant (including those of petroleum, animal, plant, or synthetic origin), a polymer, an oil, such as peanut oil, mineral oil, castor oil, soybean oil, an alcohol, a polysorbate, a sorbitan ester, an ether sulfate, a sulfate, a betaine, a glycoside, a maltoside, a fatty alcohol, a nonoxynol, a poloxamer, a polyoxyethylene, a polyethylene glycol, a dextrose, a glycerol, or a digitonine. It will be understood that the carrier will be dermatologically acceptable. A preferred carrier contains an emulsion, such as oil in water, water in oil, water in oil in water, and silicone oil in water emulsion. The emulsion may additionally contain an emulsifier and / or an antifoaming agent.

[0106] Examples of suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethyl cellulose and polyethylene glycol and any suitable binder disclosed in US2014120131 or US2004132667.

[0107] Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride and any suitable lubricant disclosed in US2014120131 or US2004132667.

[0108] The selection of pharmaceutical excipients (including carriers and diluents) may be made in relation to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable carrier(s), diluent(s), binder(s), lubricant(s), suspending agent(s), coating agent(s), and solubilizing agent(s) as, or in addition to, excipients. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0109] The composition may be formulated in a unit dosage form, that is, in the form of a unit dose, or in the form of separate parts containing multiple or sub-units of a unit dose.

[0110] A person skilled in the art can easily determine an appropriate dose of one of the compositions or peptides of the present invention for administration to a subject without excessive experimentation. Typically, a physician will determine the actual dose most suitable for an individual patient or subject, and this will depend on various factors including the activity of the specific compound used, the metabolic stability and duration of action of said compound, age, body weight, overall health, sex, diet, mode and time of administration, elimination rate, drug combination, severity of a specific condition, and the therapy the individual receives. The doses disclosed herein are examples of average cases. Of course, there may be individual cases where higher or lower dose ranges are valuable, and these are within the scope of the present invention. If necessary, the peptide / composition may be administered at a dose of 0.01 to 250 mg / kg body weight, which may be provided in one or more doses. In the embodiments, the dose is 250, 500, or 1000 mg per day regardless of body weight.

[0111] "Feed grade" means that the source is consumable and the enzymes used in its production are also safe for consumption.

[0112] As used herein, the term “expression vector of the present invention” may be any suitable vector comprising chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising a suitable set of expression control elements) suitable for the expression of the peptide of the present invention in cells. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the peptide-coding nucleic acid molecule is contained in naked DNA or RNA vectors (e.g., containing linear expression elements) (e.g., as described in the literature [Sykes and Johnston, Nat Biotech 12, 355-59 (1997)]), compact nucleic acid vectors (e.g., as described in U.S. Patent No. 30 6,077,835 and / or WO 00 / 70087), or plasmid vectors such as pBR322, pUC 19 / 18, or pUC 118 / 119. Such nucleic acid vectors and their uses are widely known in the art (e.g., see U.S. Patent No. 5,589,466 and U.S. Patent No. 5,973,972). In one embodiment, the DNA contains an expression control sequence.

[0113] In one embodiment, the vector is suitable for the expression of the peptide of the present invention in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509), pET vectors (Novagen, Madison, Wisconsin), etc. In one embodiment, the expression vector may also, or alternatively, be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase, and PGH (reviewed in [F. Ausubel et al., ed., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York]; and [Grant et al., 1987, Methods in Enzymol 153, 516-544]). In other embodiments, the expression vector is suitable for expression in baculovirus-infected insect cells (Kost, T; and Condreay, JP, 1999, Current Opinion in Biotechnology 10 (5): 428-33).

[0114] An expression control sequence is engineered to control and drive the transcription of a gene of interest and the subsequent expression of a protein in various cell systems. A plasmid combines an expressible gene of interest with an expression control sequence (i.e., an expression cassette) comprising desirable elements, such as a promoter, an enhancer, a selectable marker, an operator, etc. In the expression vector of the present invention, a peptide-coding nucleic acid molecule may include or be associated with any suitable promoter, enhancer, selectable marker, operator, repressor protein, poly-A termination sequence, and other expression-promoting elements.

[0115] As used herein, a “promoter” refers to a DNA sequence sufficient to direct the transcription of a DNA sequence that is operably linked, that is, linked in such a manner that it allows the transcription of the peptide-coding nucleotide sequence of the present invention when an appropriate signal is present. The expression of the peptide-coding nucleotide sequence may be placed under the control of any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer or CMV major IE (CMV-MIE) promoter, as well as RSV, SV40 late promoters, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters). In some embodiments, the vector comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC, and HIV LTR.

[0116] The nucleic acid molecule of the present invention can also be operably linked to an effective poly(A) termination sequence, a replication origin for an E. coli plasmid product, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker).

[0117] Nucleic acids may also include regulated inducible promoters (inducible, repressive, developmentally regulated) in contrast to constitutive promoters, such as CMV IE (a person skilled in the art will recognize that these terms are actually descriptive terms for the degree of gene expression under specific conditions).

[0118] Selectable markers are a widely known element in the relevant technical field. Under selection conditions, only cells expressing appropriate selectable markers can survive. Typically, selectable marker genes express proteins, usually enzymes, that confer resistance to various antibiotics during cell culture. Under other selection conditions, cells expressing fluorescent protein markers become visible and are therefore selectable. The embodiments include beta-lactamase (bla) (beta-lactam antibiotic resistance or ampicillin resistance gene or ampR), bls (blasticidin resistance acetyltransferase gene), bsd (blasticidin-S deaminase resistance gene), bsr (blasticidin-S resistance gene), Sh ble (Zeocin® resistance gene), hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM (tetracycline resistance gene or tetR), neomycin phosphotransferase II (npt) (neomycin resistance gene or neoR), kanR (kanamycin resistance gene), and pac (puromycin resistance gene).

[0119] In certain embodiments, the vector comprises one or more selectable marker genes selected from the group consisting of bla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM, npt, kanR 20, and pac. In other embodiments, the vector comprises one or more selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), or yellow fluorescent protein (YFP).

[0120] For the purposes of the present invention, gene expression in eukaryotic cells can be strictly regulated using a strong promoter controlled by an operator, which is consequently regulated by a regulatory protein (which may be a recombinant "regulatory fusion protein" (RFP)). The RFP essentially consists of a transcription blocking domain and a ligand-binding domain that regulates its activity. An example of such an expression system is described in US20090162901A1, the full text of which is incorporated herein by reference.

[0121] As used herein, the term “operator” refers to a DNA sequence introduced into or near a gene in such a manner that the gene can be regulated by the binding of RFP to the operator, and as a result, prevents or allows the transcription of the gene of interest, i.e., the nucleotide encoding the peptide of the present invention. A number of operators in prokaryotic cells and bacteriophages are well characterized (Neidhardt, ed., Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol 2 ASM Press, Washington DC 1996). These include, but are not limited to, the operator region of the LexA gene of E. coli that binds to the LexA peptide, and lactose and tryptophan operators that bind to repressor proteins encoded by the Lad and trpR genes of E. coli. These also include bacteriophage operators from the lambda PR and phage P22 ant / mnt genes that bind to a repressor protein encoded by lambda cI and P22 arc. In some embodiments, when the transcription blocking domain of RFP is a restriction enzyme, e.g. NotI, the operator is a recognition sequence for that enzyme. A person skilled in the art will recognize that the operator must be located adjacent to the promoter, or at the 3' end thereof, so as to be able to control transcription by the promoter. For example, U.S. Patent No. 5,972,650, incorporated herein by reference, specifies that the tetO sequence is within a certain distance from the TATA box. In specific embodiments, the operator is preferably placed immediately downstream of the promoter. In other embodiments, the operator is placed within 10 base pairs of the promoter. In an exemplary cell expression system, cells are engineered to express tetracycline repressor protein (TetR), and the protein of interest is placed under the transcriptional control of a promoter whose activity is regulated by TetR.Two tandem TetR operators (tetO) are placed immediately downstream of the CMV-MIE promoter / enhancer in the vector.

[0122] In such vectors, transcription of the gene encoding the protein of interest directed by the CMV-MIE promoter can be blocked by TetR in the absence of tetracycline or some other suitable inducer (e.g., doxycycline). In the presence of an inducer, the TetR protein cannot bind to tetO, and thus transcription followed by translation (expression) of the protein of interest occurs. (See, for example, U.S. Patent No. 7,435,553, the full text of which is incorporated herein by reference).

[0123] The vector of the present invention may also utilize a Cre-lox recombination tool to facilitate the integration of a gene of interest into the host genome. The Cre-lox strategy requires at least two of the following components: 1) a Cre recombinase, which is an enzyme that catalyzes recombination between two loxP sites; and 2) a loxP site (e.g., a specific 34-base pair sequence consisting of an 8-bp core sequence and two flanking 13-bp inverted repeats where recombination occurs) or a mutant lox site. (See, for example, [Araki et al., 1995, PNAS 92:160-4]; [Nagy, A. et al., 2000, Genesis 26:99-109]; [Araki et al., 2002, Nuc Acids Res 30(19):e103]; and US20100291626A1, all incorporated herein by reference). In another recombination strategy, a yeast-derived FLP recombinase may be used with the consensus sequence FRT (see also, for example, [Dymecki, SM, 1996, PNAS 93(12): 6191-6196]).

[0124] As used herein, the term “host cell” includes any cell suitable for expressing the recombinant nucleic acid sequence. Cells include those of prokaryotes and eukaryotes (single-cell or multi-cell), bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species, etc.), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. partoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, mammalian cells, human cells, or cell fusions such as, for example, hybridoma or quadroma. In certain embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In other embodiments, the cells are eukaryotic and are selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK21), HeLa, HepG2, WI38, MRC5, Colo25, HB 8065, HL-60, Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes, for example, retinal cells (e.g., PER.C6® cells) expressing viral genes. In some embodiments, the cell is a CHO cell. In other embodiments, the cell is a CHO K1 cell.

[0125] As used herein, the term “transformed cell of the present invention” refers to a host cell comprising a nucleic acid stably integrated into a cell genome comprising a nucleotide sequence encoding the expression of the peptide of the present invention. In another embodiment, the present invention provides a cell comprising a non-integrated (i.e., episomal) nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence encoding the expression of the peptide of the present invention. In another embodiment, the present invention provides a cell line produced by stably transfecting a host cell with a plasmid comprising an expression vector of the present invention.

[0126] The term "manipulated" as used herein and applied to cells means genetically engineered using recombinant DNA technology, and generally involves the step of synthesizing a suitable expression vector (see above) and then the step of transfecting the expression vector into a host cell (generally stable transfection).

[0127] As used herein, the term "heterogeneous expression" refers to the expression of nucleic acids in host cells that do not naturally possess nucleic acids. The insertion of nucleic acids into a heterogeneous host is performed by recombinant DNA technology.

[0128] When applied to peptides (including variants and compositions containing them), "sleep-promoting" means a peptide capable of promoting sleep in a subject. In this context, the peptide may reduce cortisol expression in cells compared to an untreated control, as determined by the assay described herein.

[0129] "Improvement in sleep" may be indicated by an increase in sleep length and a decrease in the time before sleep onset. This may be indicated by deeper or undisturbed sleep.

[0130] The term “anti-inflammatory,” as applied to the peptide (including variants), means a peptide capable of modulating the expression of one or more mediators of the inflammatory response in cells, such as macrophage migration inhibitor (MIF), VEGF, and osteopontin, RANTES, and B cell activating factor (BAFF), compared to an untreated control as determined by the assay described herein. In particular, the peptide may decrease the expression of one or more of macrophage migration inhibitor (MIF), VEGF, and osteopontin, and / or increase the expression of one or more of RANTES and B cell activating factor (BAFF).

[0131] "Inflammatory disorder" refers to an immune-mediated inflammatory state affecting humans and generally characterized by dysregulation of the expression of one or more cytokines. Examples of inflammatory disorders include skin inflammatory disorders, joint inflammatory disorders, cardiovascular inflammatory disorders, certain autoimmune diseases, lung and airway inflammatory disorders, and intestinal inflammatory disorders. Examples of skin inflammatory disorders include dermatitis, e.g., atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis. Examples of joint inflammatory disorders include rheumatoid arthritis. Examples of cardiovascular inflammatory disorders include cardiovascular disease and atherosclerosis. Examples of autoimmune diseases include type 1 diabetes, Graves' disease, Guillain-Barré disease, lupus, psoriatic arthritis, and ulcerative colitis. Examples of lung and airway inflammatory disorders include asthma, cystic fibrosis, COPD, emphysema, and acute dysphagia syndrome. Examples of intestinal inflammatory disorders include colitis and inflammatory bowel disease. Other inflammatory disorders include cancer, hay fever, periodontitis, allergies, hypersensitivity, ischemia, depression, systemic diseases, post-infectious inflammation, and bronchitis. Brief explanation of the drawing

[0132] The present invention will be more clearly understood from the following description of embodiments provided only as examples with reference to the accompanying drawings, wherein: Fig. 1: Example of absolute and relative cortisol reduction. Since the cortisol found in untreated cells would be endogenous compared to the full stimulation of cortisol release by orexin A, the difference between untreated cells and vehicle control (+ 1 μM orexin A) is used to determine the relative cortisol reduction. Fig. 2: Effect of Composition A on cortisol secretion. Composition A reduces cortisol release from H295R cells. Adrenal cortical cells were left untreated or treated with Composition A (25–200 μg / mL), an orexin inhibitor (Oxi; 10 μM), or forskolin (10 μM). Cells were stimulated with orexin A (1 μM) for 4 hours prior to the collection of cell supernatants to determine cortisol concentrations using a cortisol ELISA kit. Significance was analyzed using one-way ANOVA and outliers were removed; ** P<0.01, *** P<0.001, **** P<0.00001. Data presented are the mean ± SEM from at least three independent tests. Composition A comprises the peptide of sequence identification number: 1, the peptide of sequence identification number: 2, the peptide of sequence identification number: 3, the peptide of sequence identification number: 4, and the peptide of sequence identification number: 5. Fig. 3: Effect of the peptide of the present invention on cortisol secretion. The peptide reduced orexin-induced cortisol release from H295R cells. Adrenal cortical cells were left untreated or treated with the peptide (25 & 250 μg / mL) A) Sequence ID No.: 1, B) Sequence ID No.: 2, C) Sequence ID No.: 3, D) Sequence ID No.: 4 and E) Sequence ID No.: 5, the orexin inhibitor (Oxi; 10 μM), or forskolin (10 μM). Cells were stimulated with orexin A (1 μM) for 4 hours prior to cell supernatant being collected to determine cortisol concentrations using a cortisol ELISA kit. Significance was analyzed using one-way ANOVA; * P<0.05, ** P<0.01, **** P<0.00001. Data presented are the mean ± SEM from at least three independent tests. Fig. 4: Effect of peptide SEQ ID NO: 6 on cortisol secretion. The peptide reduced orexin-induced cortisol release from H295R cells. Adrenal cortical cells were left untreated or treated with peptide SEQ ID NO: 6 (25 & 250 μg / mL), an orexin inhibitor (Oxi; 10 μM), or forskolin (10 μM). Cells were stimulated with orexin A (1 μM) for 4 hours prior to cell supernatant collection to determine cortisol concentrations using a cortisol ELISA kit. Significance was analyzed using one-way ANOVA; * P<0.05, ** P<0.01, **** P<0.00001. Data presented are the mean ± SEM from at least three independent tests. Fig. 5: Composition A has no effect on cell viability. Adrenal cortical cells were left untreated or treated with Composition A (25–200 μg / mL), an orexin inhibitor (Oxi; 10 μM), or forskolin (10 μM) for 4 hours prior to the test in the MTT assay. The presented data are the mean ± SEM from at least three independent assays. Fig. 6: MTT assay for adrenal cortical cells using the bioactive peptides of the present invention. The peptides are not toxic to H295R cells. Percentage viability of adrenal cortical cells after treatment with A) peptide of SEQ ID NO: 1, B) peptide of SEQ ID NO: 2, C) peptide of SEQ ID NO: 3, D) peptide of SEQ ID NO: 4 & E) peptide of SEQ ID NO: 5 at 25-250 μg / mL for 4 hours. The presented data are the mean ± SEM of at least three independent experiments. Fig. 7: Proteome Profiler cytokine array. Levels of cytokine response from untreated control THP1 cells are presented in blue. Cytokine levels from cells stimulated with LPS 100 ng / ml for 24 hours are presented in green, and cytokine release from cells treated with Composition A 500 μg / ml for an additional 24 hours prior to LPS stimulation is presented in yellow. The plotted data represents the average pixel intensity from the dot plot analyzed using Image J software. Fig. 8: Effect of the peptide of the present invention on cortisol secretion. The peptide reduced orexin-induced cortisol release from H295R cells. Adrenal cortical cells were left untreated or treated with peptides (25 & 250 μg / mL) A) Sequence ID No.: 97, B) Sequence ID No.: 98, C) Sequence ID No.: 99, and D) Sequence ID No.: 100, an orexin inhibitor (Oxi; 10 μM), or forskolin (10 μM). Cells were stimulated with orexin A (1 μM) for 4 hours prior to cell supernatant collection to determine cortisol concentrations using a cortisol ELISA kit. Significance was analyzed using one-way ANOVA; * P<0.05, ** P<0.01, **** P<0.00001. Data presented are the mean ± SEM from at least three independent tests. Specific details for implementing the invention

[0133] The inventors have surprisingly discovered a number of peptides derived from brown rice (Oryza sativa) protein that have beneficial effects on sleep.

[0134] The peptide of the present invention is an antagonist of the orexin 1 receptor (OX1R) and the orexin 2 receptor (OX2R). The effect of cortisol inhibition induces a relaxed state that is helpful for sleep.

[0135] Orexin neuropeptides and their receptors were discovered in 1998 while two different research groups were searching for new signaling molecules. These peptides were discovered because they were originally thought to affect appetite (the term orexin is the Greek word for appetite, " orexisThey were named orexin-A and orexin-B (derived from). However, further studies have suggested that while orexin peptides have only a moderate effect on appetite, their effects on wakefulness and sleep are severe (Scammell and Winrow, (2011) Annual review of pharmacology and toxicology, 51, 243).

[0136] The gene for orexin and its structure are highly conserved across mammalian species, suggesting strong evolutionary pressure to maintain the structure. In mammals, orexin (A or B) binds to two subtypes of G-protein-coupled receptors (GPCRs), OX1R and OX2R, which are produced by neurons (orexin neurons) located primarily in the lateral hypothalamic region (LHA) (Soya and Sakurai, (2020) Brain Res. Mar 15; 1731:146037). Orexin receptors are usually found in the cerebral space, but they are also expressed in the adrenal glands where cortisol is synthesized. By targeting this orexin-dependent release of cortisol, the peptide of the present invention can reduce its concentration without the need to target the central nervous system (CNS).

[0137] Accordingly, particularly in aspects of the present invention, a composition (composition of the present invention) comprising at least one peptide of the present invention is provided. In a preferred embodiment, the composition comprises two peptides of the present invention. This may be a combination of any two peptides of the present invention. The composition may comprise three peptides of the present invention, four peptides, or five peptides. The composition may comprise all peptides of the present invention.

[0138] The composition is used to promote sleep in the subject.

[0139] The compositions of the present invention may be used for non-therapeutic purposes. For example, a non-therapeutic method for promoting or improving sleep in a subject is provided, comprising the step of administering a composition or a peptide of the present invention to a subject.

[0140] A non-therapeutic method for inducing relaxation in a subject is provided, comprising the step of administering a composition or peptide of the present invention to the subject.

[0141] A non-therapeutic method for reducing or preventing stress in a subject is provided, comprising the step of administering a composition or peptide of the present invention to a subject.

[0142] A non-therapeutic method for reducing cortisol in a subject is provided, comprising the step of administering a composition or peptide of the present invention to the subject.

[0143] A non-therapeutic method for promoting recovery from fatigue in a subject is provided, comprising the step of administering a composition or peptide of the present invention to a subject.

[0144] A non-therapeutic method for promoting or improving sleep in a subject is provided, comprising the step of administering a composition or peptide of the present invention to a subject.

[0145] The present invention further provides a method for treating or preventing inflammatory disorders in a subject, comprising administering a peptide or composition of the present invention to the subject.

[0146] An aspect of the present invention provides a non-therapeutic method for increasing concentration in a subject. The method comprises administering a peptide or composition of the present invention to a subject.

[0147] An aspect of the present invention provides a non-therapeutic method for improving cognitive function or ability in a subject, comprising administering a peptide or composition of the present invention to the subject. The subject may otherwise be healthy but require improvement in cognitive function due to lifestyle and / or age. In one example, the subject is menopausal or in the menopausal transition.

[0148] An aspect of the present invention provides a method for improving cognitive function or ability in a subject, comprising administering a peptide or composition of the present invention to the subject. The subject may have impaired cognitive function due to a disease or disorder that has a negative effect on cognition, such as dementia.

[0149] The subject may be a mammal, such as a human or an animal. In particular, it is a human subject. It may be any human subject of any age. It may be a subject having one or more diseases or conditions associated with a decrease in sleep, such as dementia, Alzheimer's disease, anxiety, and depression.

[0150] The subject may have one or more sleep disorders. Sleep disorders include, but are not limited to, insomnia, sleep-related breathing disorders, central hypersomnia disorders, circadian rhythm sleep-wake disorders, parasomnia, sleep-related movement disorders, and isolated symptoms.

[0151] The subject may be in an environment sensitive to stress, such as financial stress, work-related stress, social stress, education-related stress, such as exam stress, or family-related stress.

[0152] The subject may be in an environment sensitive to a lack of sufficient sleep due to lifestyle, aging, or family circumstances.

[0153] The subject may be otherwise healthy, that is, an individual that does not suffer from one or more types of sleep disorders.

[0154] The subject may be an individual with a chronic disease characterized by low energy levels, or otherwise a healthy individual without a chronic disease but with low energy due to age, trauma, or surgery.

[0155] In an embodiment of the present invention, the peptide comprises a length of about 3 to 50 amino acids, a length of about 7 to about 50 amino acids, preferably a maximum of about 10, 15, 20, 25, 30, 35, 40, 45, or 49 amino acids, preferably a maximum of about 14, 15, 16, 17, 18, 19, or 20 amino acids. This may be a maximum of 16, a maximum of 20, or a maximum of 30 amino acids.

[0156] In an embodiment of the present invention, the composition comprises (or comprises) a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 2, a peptide comprising (or consisting thereof) sequence identification number: 3, a peptide comprising (or consisting thereof) sequence identification number: 4, a peptide comprising (or consisting thereof) sequence identification number: 5, a peptide comprising (or consisting thereof) sequence identification number: 97, a peptide comprising (or consisting thereof) sequence identification number: 98, a peptide comprising (or consisting thereof) sequence identification number: 99, and a peptide comprising (or consisting thereof) sequence identification number: 100.

[0157] In an embodiment of the present invention, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 2, a peptide comprising (or consisting thereof) sequence identification number: 3, a peptide comprising (or consisting thereof) sequence identification number: 6, and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0158] In an embodiment of the present invention, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 2, a peptide comprising (or consisting thereof) sequence identification number: 3, a peptide comprising (or consisting thereof) sequence identification number: 4, a peptide comprising (or consisting thereof) sequence identification number: 5, and a peptide comprising (or consisting thereof) sequence identification number: 6.

[0159] In one embodiment, the peptide comprises (or consists of) a variant of sequence identification number: 1. This variant has a sequence having 1 to 5 amino acid changes compared to sequence identification number: 1. In one embodiment, the variant has 1 to 4 amino acid changes compared to sequence identification number: 1. In one embodiment, the variant has 1 to 3 amino acid changes compared to sequence identification number: 1. In one embodiment, the variant has 1 to 2 amino acid changes compared to sequence identification number: 1. In one embodiment, the amino acid change is a conservative amino acid change. In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion.

[0160] In one embodiment, the peptide comprises (or consists of) a variant of SEQ ID NO: 2. In one embodiment, the variant comprises a sequence having 1 to 5 amino acid changes compared to SEQ ID NO: 2. In one embodiment, the variant has 1 to 4 amino acid changes compared to SEQ ID NO: 2. In one embodiment, the variant has 1 to 3 amino acid changes compared to SEQ ID NO: 2. In one embodiment, the variant has 1 to 2 amino acid changes compared to SEQ ID NO: 2. In one embodiment, the amino acid change is a conservative amino acid change. In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion.

[0161] In one embodiment, the peptide comprises (or consists of) a variant of SEQ ID NO: 3. In one embodiment, the variant has a sequence having 1 to 5 amino acid changes compared to SEQ ID NO: 3. In one embodiment, the variant has 1 to 4 amino acid changes compared to SEQ ID NO: 3. In one embodiment, the variant has 1 to 3 amino acid changes compared to SEQ ID NO: 3. In one embodiment, the variant has 1 to 2 amino acid changes compared to SEQ ID NO: 3. In one embodiment, the amino acid change is a conservative amino acid change. In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion.

[0162] In one embodiment, the peptide comprises (or consists of) a variant of sequence identification number: 4. In one embodiment, the variant has a sequence having 1 to 5 amino acid changes compared to sequence identification number: 4. In one embodiment, the variant has 1 to 4 amino acid changes compared to sequence identification number: 4. In one embodiment, the variant has 1 to 3 amino acid changes compared to sequence identification number: 4. In one embodiment, the variant has 1 to 2 amino acid changes compared to sequence identification number: 4. In one embodiment, the amino acid change is a conservative amino acid change. In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion.

[0163] In one embodiment, the peptide comprises (or consists of) a variant of sequence identification number: 5. In one embodiment, the variant has a sequence having 1 to 5 amino acid changes compared to sequence identification number: 5. In one embodiment, the variant has 1 to 4 amino acid changes compared to sequence identification number: 5. In one embodiment, the variant has 1 to 3 amino acid changes compared to sequence identification number: 5. In one embodiment, the variant has 1 to 2 amino acid changes compared to sequence identification number: 5. In one embodiment, the amino acid change is a conservative amino acid change. In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion.

[0164] In an embodiment, the peptide comprises (or consists of) a variant of any peptide of the present invention. For example, it is a variant of a sequence selected from any one of SEQ ID NO: 97 to SEQ ID NO: 100. In one embodiment, the variant has 1 to 4 amino acid changes compared to the sequence. In one embodiment, the variant has 1 to 3 amino acid changes compared to the sequence. In one embodiment, the variant has 1 to 2 amino acid changes compared to the sequence. In one embodiment, the amino acid change is a conservative amino acid change. In one embodiment, the amino acid change is an amino acid substitution. In one embodiment, the amino acid substitution is a conservative substitution. In one embodiment, the amino acid change is an amino acid addition. In one embodiment, the amino acid change is an amino acid deletion.

[0165] Changes are selected independently. Existing changes may be the same or different at each changed location.

[0166] An example of a variant of sequence identification number: 4 is the amino acid sequence of sequence identification number: 6.

[0167] Examples of variants of sequence identification number: 1 having one amino acid change are in Table 1.

[0168]

[0169] Table 1: Sequence Identification Number: 1 variant.

[0170] Examples of variants of sequence identification number: 1 having two amino acid changes are in Table 2.

[0171]

[0172] Table 2: Sequence Identification Number: 1 variant.

[0173] Examples of variants of sequence identification number 2 having one amino acid change are in Table 3.

[0174]

[0175] Table 3: Sequence identification number: 2 variants.

[0176] Examples of variants of sequence identification number: 2 with two amino acid changes are in Table 4.

[0177]

[0178] Table 4: Sequence identification number: 2 variants.

[0179] An example of a variant of sequence identification number 2 having three amino acid changes is in Table 5.

[0180]

[0181] Table 5: Sequence identification number: 2 variants.

[0182] An example of a variant of sequence identification number 2 having four amino acid changes is in Table 6.

[0183]

[0184] Table 6: Sequence identification number: 2 variants.

[0185] An example of a variant of sequence identification number: 3 having one amino acid change is in Table 7.

[0186]

[0187] Table 7: Sequence identification number: 3 variants.

[0188] An example of a variant of sequence identification number 3 with two amino acid changes is in Table 8.

[0189]

[0190] Table 8: Sequence identification number: 3 variants.

[0191] An example of a variant of sequence identification number: 3 with three amino acid changes is in Table 9.

[0192]

[0193] Table 9: Sequence identification number: 3 variants.

[0194] An example of a variant of sequence identification number: 3 with four amino acid changes is in Table 10.

[0195]

[0196] Table 10: Sequence identification number: 3 variants.

[0197] An example of a variant of sequence identification number: 3 with 5 amino acid changes is in Table 11.

[0198]

[0199] Table 11: Sequence identification number: 3 variants.

[0200] An example of a variant of sequence identification number: 4 having one amino acid change is in Table 12.

[0201]

[0202] Table 12: Sequence identification number: 4 variants.

[0203] An example of a variant of sequence identification number: 4 having two amino acid changes is in Table 13.

[0204]

[0205] Table 13: Sequence identification number: 4 variants.

[0206] An example of a variant of sequence identification number: 4 with three amino acid changes is in Table 14.

[0207]

[0208] Table 14: Sequence identification number: 4 variants.

[0209] An example of a variant of sequence identification number: 5 having one amino acid change is in Table 15.

[0210]

[0211] Table 15: Sequence identification number: 5 variant.

[0212] An example of a variant of sequence identification number: 5 having two amino acid changes is in Table 16.

[0213]

[0214] Table 16: Sequence identification number: 5 variant.

[0215] An example of a variant of sequence identification number: 5 having three amino acid changes is in Table 17.

[0216]

[0217] Table 17: Sequence identification number: 5 variants.

[0218] Variants of sequence identification number: 97 include the following:

[0219] QLLSQST (Sequence ID: 101)

[0220] QLLSQSTSQW (Sequence ID: 102)

[0221] QLLSQSTSQ(Sequence ID: 103)

[0222] Variants of sequence identification number: 98 include the following:

[0223] QQQYYPGFSN (Sequence Identification Number: 104)

[0224] QQQYYPGLSN (Sequence Identification Number: 105)

[0225] FQQQYYPGL (Sequence ID: 106)

[0226] QQYYPGLS (Sequence Identification Number: 107)

[0227] FQQQYYPGLS (Sequence ID: 108)

[0228] QQQYYPGLSNE (Sequence ID: 109)

[0229] FQQQYYPGLSN (Sequence Identification Number: 110)

[0230] QQYYPGLSN (Sequence Identification Number: 111)

[0231] QQQYYPGL (Sequence ID: 112)

[0232] QQYYPGLSNE (Sequence ID: 113)

[0233] The variant of sequence identification number: 99 includes the following:

[0234] ADTYNPRA (Sequence ID: 114)

[0235] LADTYNPRA (Sequence ID: 115)

[0236] PNLADTYNPR (Sequence ID: 116)

[0237] NLADTYNPRA (Sequence ID: 117)

[0238] LADTYNP (Sequence ID: 118)

[0239] ADTYNPR (Sequence ID: 119)

[0240] LADTYNPRAG (Sequence ID: 120)

[0241] RADTYNPR (Sequence ID: 121)

[0242] NLADTYNPR (Sequence ID: 122)

[0243] HADTYNPR (Sequence ID: 123)

[0244] Variants of sequence identification number: 100 include the following:

[0245] IGGIGTVPVGRV (Sequence ID: 124)

[0246] YKIGGIGTVPV (Sequence ID: 125)

[0247] IGGVGTVPVGK (Sequence Identification Number: 126)

[0248] KIGGIGTVPV (Sequence Identification Number: 127)

[0249] IGGIGTVPV (Sequence ID: 128)

[0250] GIGTVPVGRV (Sequence ID: 129)

[0251] GGIGTVPVGR (Sequence ID: 130)

[0252] IGGIGTVPVGRVE (Sequence ID: 131)

[0253] YKIGGIGTVPVGR (Sequence ID: 132)

[0254] GIGTVPVGRVE (Sequence ID: 133)

[0255] KIGGIGTVPVGR (Sequence ID: 134)

[0256] IGGIGTVPVG (Sequence ID: 135)

[0257] KIGGIGTVPVG (Sequence Identification Number: 136)

[0258] YKIGGIGTVPVG (Sequence Identification Number: 137)

[0259] GIGTVPVGR (Sequence ID: 138)

[0260] GGIGTVPVGRVE (Sequence ID: 139)

[0261] In an embodiment, the composition comprises substantially all peptides of the present invention. In one embodiment, the composition comprises substantially all variants of the present invention. In one embodiment, the composition contains substantially no other peptides.

[0262] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1 and a peptide comprising (or consisting thereof) SEQ ID NO: 2. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1 and a peptide comprising (or consisting thereof) SEQ ID NO: 3. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1 and a peptide comprising (or consisting thereof) SEQ ID NO: 4. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1 and a peptide comprising (or consisting thereof) SEQ ID NO: 5.

[0263] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 2 and a peptide comprising (or consisting thereof) SEQ ID NO: 3. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 2 and a peptide comprising (or consisting thereof) SEQ ID NO: 4. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 2 and a peptide comprising (or consisting thereof) SEQ ID NO: 5.

[0264] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 3 and a peptide comprising (or consisting thereof) sequence identification number: 4. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 3 and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0265] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 4 and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0266] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1, a peptide comprising (or consisting thereof) SEQ ID NO: 2, and a peptide comprising (or consisting thereof) SEQ ID NO: 3. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1, a peptide comprising (or consisting thereof) SEQ ID NO: 2, and a peptide comprising (or consisting thereof) SEQ ID NO: 4. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1, a peptide comprising (or consisting thereof) SEQ ID NO: 2, and a peptide comprising (or consisting thereof) SEQ ID NO: 5.

[0267] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1, a peptide comprising (or consisting thereof) SEQ ID NO: 3, and a peptide comprising (or consisting thereof) SEQ ID NO: 4. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 1, a peptide comprising (or consisting thereof) SEQ ID NO: 3, and a peptide comprising (or consisting thereof) SEQ ID NO: 5.

[0268] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 4, and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0269] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 2, a peptide comprising (or consisting thereof) SEQ ID NO: 3, and a peptide comprising (or consisting thereof) SEQ ID NO: 4. In an embodiment, the composition comprises a peptide comprising (or consisting thereof) SEQ ID NO: 2, a peptide comprising (or consisting thereof) SEQ ID NO: 3, and a peptide comprising (or consisting thereof) SEQ ID NO: 5.

[0270] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 2, a peptide comprising (or consisting thereof) sequence identification number: 4, and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0271] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 3, a peptide comprising (or consisting thereof) sequence identification number: 4, and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0272] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 2, a peptide comprising (or consisting thereof) sequence identification number: 3, and a peptide comprising (or consisting thereof) sequence identification number: 5.

[0273] In an embodiment, the composition comprises one or more peptides selected from a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 3, and a peptide comprising (or consisting thereof) sequence identification number: 100.

[0274] In an embodiment, the composition comprises a peptide comprising (or consisting thereof) sequence identification number: 1, a peptide comprising (or consisting thereof) sequence identification number: 3, and a peptide comprising (or consisting thereof) sequence identification number: 100.

[0275] In each of these embodiments, the peptide may include a variant of the sequence.

[0276] It will be recognized that the composition may include any combination of the peptides of the present invention.

[0277] In an embodiment, the composition comprises one or more peptides selected from peptides comprising (or consisting thereof) a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100. In an embodiment, the composition comprises all peptides selected from peptides comprising (or consisting thereof) a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100.

[0278] Preferably, the composition may be for oral administration. For example, as a dietary supplement or a nutritional supplement. It will be recognized that this may be any suitable form.

[0279] The peptide of the present invention can be used in supplements or pharmaceutical products to promote sleep.

[0280] The peptides or compositions of the present invention may be modified for local, oral, rectal, parenteral, intramuscular, intraperitoneal, intra-arterial, intrabronchial, subcutaneous, intradermal, intravenous, nasal, vaginal, buccal, or sublingual administration routes. The compositions of the present invention may be presented, prepared, or administered in various suitable forms. Such forms include, but are not limited to, liquid, semi-solid, and solid administration forms, such as liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, emulsions, microemulsions, tablets, pills, powders, liposomes, dendrimers, and other nanoparticles, microparticles, capsules, suppositories, and tablets. It will be recognized that the form may depend on the intended mode of administration, the characteristics of the composition or combination, and the therapeutic application or other intended use. The formulation may also include, for example, powder, paste, ointment, jelly, wax, oil, lipid, lipid (cationic or anionic) containing vesicle, DNA conjugate, anhydrous absorbent paste, oil in water and water in oil emulsions, emulsions, carbowax (polyethylene glycol of various molecular weights), semi-solid gel, and semi-solid mixture containing carbowax.

[0281] In some embodiments of the present invention, the composition may be delivered through any one of a microemulsion and nanoemulsion microsphere, microparticle, or microparticle, including a liposome, mixed liposome, oleosome, niosome, ethosome, millicapsule, capsule, macrocapsule, nanocapsule, nanostructured lipid carrier, sponge, cyclodextrin, vesicle, micelle, mixed micelle of surfactant, surfactant-phospholipid mixed micelle, millisphere, sphere, lipid sphere, particle, nanosphere, nanoparticle, milliparticle, solid nanoparticle, as well as a water-in-oil microemulsion having an internal structure of an inverse micelle.

[0282] Various methods are available for manufacturing liposomes. For example, the literature [Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980)], U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91 / 17424, [Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976)]; [Fraley, et al., PNAS 76:3348-3352 (1979)]; [Hope et al., Biochim. Biophys. Acta 812:55-65 (1985)]; [Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986)]; [Williams et al., PNAS 85:242-246 (1988)]; [Liposomes (Ostro (ed.), 1983, Chapter 1)]; [Hope et al., Chem. Phys. Lip. 40:89 (1986)]; See [Gregoriadis, Liposome Technology (1984)] and [Lasic, Liposomes: from Physics to Applications (1993)]. Suitable methods include, for example, sonication, extrusion, high pressure / homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposomal vesicles, and ether fusion methods, all of which are widely known in the relevant art.

[0283] These delivery systems may be modified to achieve greater penetration of the compounds and / or peptides of the present invention. This may improve pharmacokinetic and pharmacodynamic properties. The delivery system may be a sustained-release system in which the compounds or peptides of the present invention are gradually released at a constant release rate over a period of time, and preferably over a period of time. The delivery system is manufactured by methods known in the relevant art. The amount of peptide contained in the sustained-release system will depend on the location where the composition is to be delivered and the duration of release, as well as the type of condition, disease, and / or disorder to be treated or cured.

[0284] In embodiments of the present invention, the composition may further comprise at least one pharmaceutically acceptable excipient. The excipient may be used interchangeably with the functional component or additive. Although the peptide or composition of the present invention may be administered alone, it will be understood that they will generally be administered in combination with pharmaceutical excipients. Pharmaceutically acceptable excipients are any known excipients that are widely known in the relevant art. Examples of suitable excipients are disclosed herein.

[0285] Preferably, any excipients included are present in trace amounts. The amount of excipients included will depend on numerous factors, including the type of excipient used, the characteristics of the excipient, the amount of the active ingredient or peptide in the composition, and / or the intended use of the composition. The characteristics and amount of any excipients should not impermissibly alter the benefits of the peptide of the present invention.

[0286] In embodiments of the present invention, the composition may further comprise one or more additional components. The composition of the present invention may be administered sequentially, simultaneously, or sequentially with one or more other additional agents. These additional components may be beneficial to include in a composition formulated for oral administration in a specific manner, for example, or may be beneficial depending on the intended use of the composition, for example, for promoting sleep. The additional components may be active, functional, or both.

[0287] Advantageously, the peptide or composition may be used as a sole means of promoting and supporting restful sleep, or in combination with other similarly indicated compositions or agents, or as a component of a larger composition.

[0288] In certain embodiments, the peptide or composition must be administered in combination with one or more other active agents, for example, commercially available conventional anti-inflammatory agents, conventional sleep promoters, or pharmacological enhancers. In such cases, the peptide(s) or composition of the present invention may be administered sequentially, simultaneously, or sequentially with one or more other active agents.

[0289] It will be recognized that multiple additional ingredients may be added. The amount of additional ingredients may be about 0.001 wt% to about 50 wt% of the composition, preferably about 0.01 wt% to about 20 wt%, preferably about 0.1 wt% to about 10 wt%, about 0.5 wt% to about 10 wt%, about 1 wt% to about 5 wt%, preferably 2 wt%. The amount of additional ingredients included will depend on numerous factors, including the type of additional ingredient used, the characteristics of the additional ingredient, the component(s) of the composition, the amount of the active ingredient or peptide in the composition, and / or the intended use of the composition. The characteristics and amount of any additional ingredients should not alter the benefits of the peptide of the present invention to an unacceptable degree.

[0290] The composition of the present invention may be alcohol-free.

[0291] The peptide of the present invention is used in the composition of the present invention at a therapeutically effective concentration to achieve the desired effect; in a preferred form relative to the total weight of the composition, 0.00000001% (weight) to 20% (weight); preferably 0.000001% (weight) to 15% (weight), more preferably 0.0001% (weight) to 10% (weight), and even more preferably 0.0001% (weight) to 5% (weight). Ideally, the peptide of the present invention is used in an amount of about 0.00001% w / w to about 0.5% w / w [0.1 to 5000 ppm] of the composition, more preferably 0.00005 w / w to about 0.05 w / w [0.5 to 500 ppm], and most preferably about 0.0001 w / w to about 0.01 w / w [1 to 100 ppm]. Ideally, the peptide of the present invention is preferably used in an amount of about 0.0001% w / w to about 0.004% w / w of the composition.

[0292] It must be understood that an ingredient considered to be an "active" ingredient in one product may be a "functional" or "excipient" ingredient in another, and vice versa. Furthermore, it will be recognized that some ingredients serve a dual role, acting as both an active ingredient and a functional or excipient ingredient.

[0293] In a preferred embodiment, repeated use of the peptide(s) or composition is provided.

[0294] The present invention also includes a food comprising the peptide of the present invention or the composition of the present invention. In a preferred embodiment, the food is intended for human consumption.

[0295] The composition of the present invention may be a food.

[0296] In one embodiment, the edible product is a beverage. In one embodiment, the edible product is a bakery product. In one embodiment, the edible product is a dairy product. In one embodiment, the edible product is a snack product. In one embodiment, the edible product is a baked extruded food. In one embodiment, the edible product is powdered milk. In one embodiment, the edible product is an infant formula. In one embodiment, the edible product is a confectionery product. In one embodiment, the edible product is yogurt. In one embodiment, the edible product is a yogurt beverage. In one embodiment, the edible product is an ice cream product. In one embodiment, the edible product is a frozen food. In one embodiment, the edible product is a breakfast cereal. In one embodiment, the edible product is bread. In one embodiment, the edible product is a flavored milk beverage. In one embodiment, the edible product is a confectionery bar. In one embodiment, the edible product is tea or a tea product. In one embodiment, the edible product is a baked extruded snack product. In one embodiment, the edible product is a fried snack product. In one embodiment, the edible product is a nutritional supplement. In one embodiment, the edible product is a sports nutrition product. In one embodiment, the edible product is infant food. In one embodiment, the edible product is a special food for immunocompromised individuals. In one embodiment, the edible product is a food for elderly patients.

[0297] In one embodiment, the composition constitutes 0.001 to 1.0% (w / w) of the food. In one embodiment, the composition constitutes 0.005 to 0.5% (w / w) of the food. In one embodiment, the composition constitutes 0.005 to 0.1% (w / w) of the food. In one embodiment, the composition constitutes 0.01 to 0.03% (w / w) of the food. In one embodiment, the composition constitutes about 0.02% (w / w) of the food.

[0298] The composition may be provided in a solid dosage form having specific controlled release characteristics. This may be in a layered dosage form, and each layer has a unique release or dissolution characteristic.

[0299] The present invention will now be described with reference to specific embodiments. These are merely illustrative and for illustrative purposes only: they are not intended to limit the scope of the claimed proprietary rights or the described invention in any way. These embodiments constitute the best manner currently considered for practicing the present invention.

[0300] Examples

[0301] Peptides and compositions used

[0302] The peptides used are the following peptides: sequence identification number: 1, sequence identification number: 2, sequence identification number: 3, sequence identification number: 4, sequence identification number: 5, sequence identification number: 6, sequence identification number: 97, sequence identification number: 98, sequence identification number: 99, and sequence identification number: 100.

[0303] Composition A is a combination of sequence identification number: 1, sequence identification number: 2, sequence identification number: 3, sequence identification number: 4, and sequence identification number: 5.

[0304] Peptide characterization

[0305] Mass Spectroscopic Analysis

[0306] A sample volume containing 5 mg of peptide was acidified with 0.2% formic acid (Sigma-Aldrich, St. Louis, Missouri, USA), desalted, and concentrated using an Oasis HLB Prime SPE cartridge (Waters Corporation, Milford, Massachusetts, USA). The eluent was lyophilized and resuspended in 100 μL of Optima Grade LC / MS water (Fisherbrand, Thermo Fisher Scientific Inc., Canoga Park, California, USA). Peptide content was determined using the BCA assay. An aliquot containing 20 μg is resuspended in 0.1% TFA (Sigma-Aldrich, St. Louis, Missouri, USA) containing a Pierce™ peptide retention time calibration mixture at a final concentration of 1 mg / mL (Thermo Fisher Scientific Inc., Canoga Park, California, USA).

[0307] Samples were analyzed by a nano LC-MS / MS Dionex Ultimate 3000 coupled to a Thermo Fisher Q Exactive (Thermo Fisher Scientific Inc., Canoga Park, California, USA) in bipolar mode. Peptides were loaded onto a trapping column and eluted on a 25 cm analytical column PepMap RSLC C18 (Thermo Fisher Scientific Inc., Canoga Park, California, USA) with a 1 h gradient at a flow rate of 300 nL / min. The mass spectrometer operated in a data-dependent manner, and MS and MS / MS were performed on an Orbitrap with resolutions of 70,000 FWHM and 17,500 FWHM, respectively. From the MS scans, the 15 most intense ions were selected for MS / MS.

[0308] H295R cell culture

[0309] Adrenal cortical cells, NCI-H295R [H295R] (American Type Culture Collection, ATCC-CRL-2128™, Rockville, Maryland) were maintained in culture using Gibco Dulbecco Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F-12) (Thermo Fisher Scientific) supplemented with Corning's 1% ITS+ Premix (Catalog No. 354352), Corning's 2.5% Nu-Serum (Catalog No. 355100), and 1% Penicillin-Streptomycin, in accordance with the manufacturer's guidelines. Cells were maintained viable at 37°C and 5% CO2.

[0310] Cortisol ELISA

[0311] H295R cells (300,000 cells / mL) were seeded into 24-well plates for 72 hours. After this time, non-adherent cells were aspirated, and adherent cells were serum depleted for an additional 24 hours. After depletion, cells were treated with the relevant assay control and peptide or composition (3.25–200 μg / mL) or remained untreated.

[0312] A series of assay controls were used to validate the assay. Cells were stimulated to release cortisol using 1 μM orexin A (OxA; natural ligand of the orexin receptor; O6012) (Sigma) or 10 μM forskolin (F6886) (Sigma), while 10 μM orexin inhibitor (Oxi) SB-334867 (SML1530) (Sigma) was used to inhibit cortisol release (as assay controls alone, not commercially available). All peptide and NPN treatments were performed in serum-free medium containing 1 μM orexin A. Cells were treated for 4 hours prior to the collection of cell supernatants to determine cortisol concentrations using a commercially available ELISA kit (Cortisol ELISA Kit, ab154996, Abcam) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate spectrophotometer (CLARIOstar BMGlabtech). Data were expressed as inhibition of orexin-induced concentrations of cortisol.

[0313] Toxicity testing

[0314] The MTT assay was used to determine whether the peptide was toxic to the cells upon treatment. H295R cells were seeded into 24-well plates (150,000 cells / well) and treated with Composition A (100–200 μg / mL). H295R cells were treated for 4 hours before the well contents were removed and replaced with MTT reagent (0.5 mg / mL). The cells were treated with MTT reagent for 2 hours at 37°C, 95% humidity, and 5% CO2. The MTT reagent was removed from the cells and replaced with 100% DMSO for 5 minutes while shaking on a plate shaker. The plates were read at 570 nm using a ClarioStar BMG LabTech plate reader. The generated optical density values ​​were converted to %, with untreated cells set to 100%.

[0315] Visualization and Statistics

[0316] All graphs and statistical analyses were performed using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San Diego, California, USA). All treatment groups were normalized relative to the vehicle control group, and all were treated with orexin A. Where significant differences between the treatment groups and the vehicle control group were appropriate, they were determined by one-way ANOVA followed by the Dunnet test or Kruskal-Wallis test followed by the Dunnet test. Association analyses are indicated in the figure legend. Data are presented as a percentage of the untreated control group (mean ± SEM of at least three independent experiments unless otherwise indicated). For all analyses, a P value < 0.05 was considered significant. The peptide effect on cortisol secretion is calculated as absolute cortisol reduction (amount of % reduction in cortisol compared to the vehicle control group) or relative % reduction in cortisol (Fig. 1). Here, the difference in cortisol secretion between the untreated and vehicle control groups is known as relative cortisol reduction and represents the maximum amount of required cortisol reduction, at which point the released cortisol will return to baseline levels.

[0317] Preclinical safety study

[0318] Simulated in vitro peptide stability

[0319] For key components of functional ingredients that modulate downstream physiological pathways, they must survive intestinal passage, and depending on their target, they may need to be absorbed across the intestinal barrier. Bioavailability and stability can be key to promoting the adoption of functional ingredients as both preventive and interventional nutritional strategies, not only by identifying the active components of the functional ingredients but also by subsequently evaluating their efficacy. The characterization of the peptide of the present invention is performed by identifying the constituent active molecules, subsequently verifying their efficacy, and evaluating bioavailability and stability in a biological matrix. In this experiment, the inventors synthetically reproduced and verified the effects of the bioactive peptide of the present invention. The bioaccessibility and bioavailability of the peptide were measured by evaluating in vitro the predicted ability of the peptide to mimic upper intestinal passage and be transported across the intestinal barrier.

[0320] Anti-inflammatory research

[0321] Cytokine array

[0322] The Proteome Profiler Human XL Cytokine Array Kit (R&D Systems), containing four pre-coated nitrocellulose membranes with 105 duplicate cytokine and chemokine markers, was used to identify various inflammatory cytokines that could be secreted in response to Composition A on THP-1 cells. The supernatant was collected from THP-1 cells and added to pre-coated and pre-blocked nitrocellulose blots containing 15 μL of detection antibody for 1 hour, and incubated overnight on a vibrating platform at 4°C. The membranes were then washed three times with 1x wash buffer for 30 minutes at room temperature prior to the addition of HRP solution. The blots were re-washed and blotted dry prior to examination with a chemiluminescent reagent for imaging in a G-box. Densitometric analysis was used to evaluate cytokine expression and release. Three pairs of reference markers dotted on the nitrocellulose blot verify the validity of the test. The data were presented as a percentage (%) area in terms of pixelation.

[0323] result

[0324] Characterization

[0325] Table 18 below illustrates the results of peptide characterization.

[0326]

[0327] Table 18: Peptide Characterization. This table presents the sequence, length, molecular weight, and charge of the peptides of the present invention.

[0328] Pre- & non-clinical research

[0329] In vitro data analysis and results

[0330] Antagonistic orexin signaling pathway and reduced cortisol secretion

[0331] Composition A exhibited a dose-response to cortisol release, with treatment at 200 μg / mL inducing the greatest effect, measuring an absolute reduction of 17.4% and an induced reduction of 44.3% (Fig. 2). Orexin A is a natural ligand and agonist of the orexin signaling pathway; accordingly, it was used as a vehicle control for all tested samples, except for the untreated ones, which allows for a clear distinction between unstimulated and stimulated cells. Two additional assay controls were used to validate the assay: an orexin inhibitor (Oxi; 10 μM) as a positive control for inhibition and forskolin (10 μM) as a control for activation. Since Oxi inhibited cortisol to levels lower than those observed endogenously, it also inhibited non-orexin-induced cortisol. However, because it also potently inhibited orexin-induced cortisol, it was considered an appropriate control (Fig. 2). Samples capable of modulating cortisol to endogenous concentrations in the presence of orexin ligand were considered positive results, which is one of the reasons for calculating not only induced cortisol release but also absolutes. Forskolin was used to verify cortisol release from the adrenal cell lines used in the test.

[0332] Composition A is a peptide matrix, but the activity of the peptide predicted to yield anti-orexin activity was characterized. The peptide was synthetically synthesized and tested for cortisol modulation in H295R cells.

[0333] Synthesized and in vitro Five tested peptides were shown to be active. After 4-hour incubation, sequence number 1 (Fig. 3A), sequence number 2 (Fig. 3B), sequence number 3 (Fig. 3C), and sequence number 4 (Fig. 3D) significantly inhibited cortisol release at 250 µg / mL (* P<0.05), with absolute reductions of 14%, 10%, 19%, and 8%, respectively. This is equivalent to relative reductions of 30%, 21%, 54%, and 17% in endogenous cortisol secretion. However, sequence identification number: 4 (Fig. 3D) and sequence identification number: 5 (Fig. 3E) significantly inhibited cortisol release at a lower concentration of 25 µg / mL, with absolute reductions of 17% and 13% recorded, respectively, which are equivalent to relative reductions of 36% and 27%. The absolute reductions observed for the individual peptides were smaller than those for composition A and occurred at much higher concentrations. This suggests that there is a possibility of synergistic or additive effects when the peptides are combined.

[0334] Four additional peptides were tested in vitro and were also shown to possess activity. Refer to Figures 8(A) through (D) for the peptides of sequence identification number: 97, sequence identification number: 98, sequence identification number: 9, and sequence identification number: 100. In Figures 8A and B, sequence identification number: 97 and sequence identification number: 98 significantly inhibited cortisol release at 25 (* P<0.05; *** P<0.001, respectively) and 250 µg / mL (**** P<0.00001, respectively). In Figure 8C, sequence identification number: 99 significantly inhibited cortisol release at 250 µg / mL (* P<0.05). In Figure 8D, sequence identification number: 100 significantly inhibited cortisol release at 25 µg / mL (* P<0.05). These results suggest that these peptides contribute to the effect on cortisol production observed with the application of composition A.

[0335] Composition A and cell viability

[0336] The viability of adrenal cortical cells was not affected in the MTT assay after 4 hours of treatment with an orexin inhibitor (Oxi; 10 μM), forskolin (10 μM), or composition A (25–200 μg / mL) (Figs. 5, 6). Untreated cells were considered 100% viable, and therefore the effect on cell viability in treated cells was calculated as a percentage of untreated control cells. This data suggests that composition A did not have any adverse effects on in vitro cell viability within the dose range used.

[0337] Preclinical in vitro experiment with Composition A

[0338] The peptide in composition A survives in in vitro simulated gastrointestinal digestion and crosses the epithelial layer.

[0339] Gastrointestinal digestion analysis and Transwell absorption assay

[0340] The stability of Composition A was evaluated in vitro through the upper intestinal tract, consisting of a 2-hour gastric phase followed by a 2-hour intestinal phase. It was shown that the peptides of sequence identification numbers 1, 2, 4, and 5 survived the proteolytic effects of pepsin, acidic gastric conditions, and subsequently, 2-hour simulated intestinal digestion (Table x). The intact peptide pep_3KDR0P was not detected after the gastric phase, suggesting that it may have been cleaved into a shorter sequence. Depending on where this cleaving occurred, it may retain its bioactivity.

[0341] The bioavailability of the peptides was tested by treating the Caco-2:HT29 intestinal barrier with S-GID composition A using an FDA-approved technique for measuring human intestinal permeability (Li et al (2016) Planta Med. 82, 1202-7; Larregieu (2013) AAPS J. 15, 483-97). The integrity of the intestinal barrier was maintained throughout the study, as TEER values ​​did not change significantly before and after treatment and were not different from the negative control (cells containing medium, data not shown). Interestingly, three peptides (SEQ ID: 1, SEQ ID: 2, and SEQ ID: 4) were able to cross the intestinal co-culture and thus have the potential to reach downstream target organs (Table 19). This also forms the basis for the fact that these peptides are resistant to the metabolic activity of gastric and intestinal proteases, as well as pineal margin enzymes such as aminopeptidase, endopeptidase, and carboxypeptidase.

[0342]

[0343] Table 19: Summary of peptides in Composition A surviving in S-GID and Transwell uptake assays.

[0344] Anti-inflammatory research

[0345] Cytokine array

[0346] The Proteome Profiler Human XL Cytokine Array Kit was used to measure the secretion of various cytokines from THP1 cells for an additional 24 hours, either untreated or pre-treated with 500 μg / ml of Composition A for 24 hours prior to stimulation with 100 ng / ml LPS. This array provides a high-throughput screening approach to investigate potential cytokines and targets that the compositions of the present invention can modulate. Figure 7 provides a visual indication of the stimulating effect of LPS on activating pro-inflammatory pathways in THP1 cells compared to untreated cells. Furthermore, this highlights the modulating effect of Composition A on a subset of proteins, where cytokine expression is similar to that of untreated cells. Among the 105 human proteins contained in the kit, Composition A was shown to modulate the expression of 13 proteins in particular. Among these are known factors in inflammation associated with sleep apnea, which are critically involved in immune function during anxiety, depression, and stress. Macrophage migration inhibitor (MIF) ; having a role in hippocampal neurogenesis and response to stress VEGF ; inducing different white blood cells and inducing the secretion of inflammation-inducing cytokines, Osteopontin ; Promoting the mobilization and activation of inflammatory monocytes, lymphocytes, and mast cells RANTES ; and inflammatory cytokines involved in the regulation of both innate and adaptive immune responses, B cell activation factor (BAFF) There are important mediators of the inflammatory response, including

[0347] argument

[0348] High levels of stress in daily life are causing poor-quality sleep for millions of people worldwide. There is concern regarding the cascading effects this can have on physical, mental, and social health. Prescribed sleep medications fall short of addressing this growing problem because the unconscious alone does not trigger cell repair, tissue restoration, memory consolidation, or many of the other important biological processes that occur during the natural, healthy stages of sleep. While natural sleep remedies have been used for centuries, their safety and efficacy are debated, and their mechanisms of action and molecular targets are generally not known.

[0349] The inventors identified a bioactive peptide within the proteome of Oryza sativa that induces calmness and promotes healthy sleep.

[0350] Peptides were characterized using MS and tested to confirm their biological activity. The peptides of the present invention were characterized for orexin antagonist activity and anti-inflammatory properties, as well as potential anti-anxiety properties. Crucially, these peptides do not exert a detectable effect on cell viability. Furthermore, the peptides were not only able to survive in simulated gastrointestinal digestion but were also able to traverse the intestinal epithelial layer in vitro.

[0351] The present invention not only significantly increases cortisol secretion from adrenal cortical cells but also exerts anti-inflammatory effects. These effects have been confirmed in vitro.

[0352] Equivalents

[0353] The foregoing description details preferred embodiments of the present invention. Considering these descriptions, it is expected that numerous variations and modifications will occur in the practice to those skilled in the art. Such variations and modifications are intended to be included within the scope of the claims appended herein.

Claims

Claim 1 A peptide of up to 50 amino acids in length comprising an amino acid sequence selected from sequence identification numbers: 1 to 5 and 97 to 100 or an effective variant of an amino acid sequence. Claim 2 A peptide according to claim 1, wherein the variant comprises one to three amino acid changes compared to the sequence, wherein each amino acid change is independently selected from deletion, addition, substitution, and insertion. Claim 3 A peptide that is a sleep-promoting peptide in paragraph 1 or 2. Claim 4 A peptide that is an anti-inflammatory peptide and / or an anti-anxiety peptide in any one of paragraphs 1 to 3. Claim 5 A peptide according to any one of claims 1 to 4, wherein the variant comprises an amino acid sequence selected from sequence identification numbers: 6 to 96 and 101 to 139. Claim 6 A modified peptide according to any one of claims 1 to 5, preferably to increase the stability of the peptide. Claim 7 A conjugate comprising one or more peptides of any one of claims 1 to 6, joined, connected, or fused to a binding partner. Claim 8 A composition comprising one or more peptides of any one of claims 1 to 6 or a conjugate of claim 7. Claim 9 A composition according to claim 8, comprising a peptide or an effective variant thereof comprising sequence identification number: 1, a peptide or an effective variant thereof comprising sequence identification number: 2, a peptide or an effective variant thereof comprising sequence identification number: 3, a peptide or an effective variant thereof comprising sequence identification number: 4, and a peptide or an effective variant thereof comprising sequence identification number:

5. Claim 10 A composition according to claim 8, comprising a peptide consisting of sequence identification number: 1, a peptide consisting of sequence identification number: 2, a peptide consisting of sequence identification number: 3, a peptide consisting of sequence identification number: 4, and a peptide consisting of sequence identification number:

5. Claim 11 In claim 8, a composition comprising a peptide comprising sequence identification number: 1 or an effective variant thereof, a peptide comprising sequence identification number: 3 or an effective variant thereof, and / or a peptide comprising sequence identification number: 100 or an effective variant thereof. Claim 12 A composition according to claim 11 comprising a peptide consisting of sequence identification number: 1, a peptide consisting of sequence identification number: 3, and a peptide consisting of sequence identification number:

100. Claim 13 A composition that is a powder in any one of paragraphs 8 to 12. Claim 14 A nutritional or dietary supplement comprising a peptide of any one of claims 1 to 6, a conjugate of claim 7, or a composition of any one of claims 8 to 12. Claim 15 A peptide, conjugate, composition, or supplement for use in promoting sleep in a subject in any one of claims 1 to 13 and 114. Claim 16 A non-therapeutic method for promoting sleep in a subject, comprising administering to the subject a peptide of any one of claims 1 to 6, a conjugate of claim 7, a composition of any one of claims 8 to 13, or a supplement of claim 14. Claim 17 A peptide, conjugate, composition, or supplement of the present invention for use in reducing cortisol in a subject, in any one of claims 1 to 14. Claim 18 A peptide, conjugate, composition, or supplement of the present invention for use in treating or preventing stress or anxiety in a subject, in any one of claims 1 to 14. Claim 19 A non-therapeutic method for reducing fatigue in a subject, comprising administering to the subject a peptide of any one of claims 1 to 6, a conjugate of claim 7, a composition of any one of claims 8 to 13, or a supplement of claim 14. Claim 20 A peptide, conjugate, composition, or supplement for use in the prevention or treatment of inflammatory disorders in a subject, in any one of claims 1 to 14. Claim 21 A non-therapeutic method for reducing inflammation in a subject, comprising administering to the subject a peptide of any one of claims 1 to 6, a conjugate of claim 7, a composition of any one of claims 8 to 13, or a supplement of claim 14. Claim 22 A non-therapeutic method for increasing concentration in a subject, comprising administering to a subject a peptide of any one of claims 1 to 6, a conjugate of claim 7, a composition of any one of claims 8 to 13, or a supplement of claim 14. Claim 23 A non-therapeutic method for improving cognitive function or ability in a subject, comprising the administration of a peptide of any one of claims 1 to 6, a conjugate of claim 7, a composition of any one of claims 8 to 13, or a supplement of claim 14 to a subject. Claim 24 A peptide, conjugate, composition, or supplement according to any one of claims 1 to 14, intended for use in preventing cognitive decline or improving cognitive function in a subject, wherein the subject has a disorder or disease that has a negative effect on cognition.

Images